Immunogenic composition

ABSTRACT

The present invention relates to immunogenic compositions comprising a dried solid or highly viscous liquid formulation of inactivated polio virus (IPV) and a stabilizing agent wherein the IPV retains its antigenicity and/or immunogenicity. Methods of producing a dried formulation of IPV which retains its antigenicity/immunogenicity are described.

This application is a 371 of International Application No.PCT/EP2003/012160, filed 30 Oct. 2003.

The present invention relates to immunogenic compositions comprising adried solid or high viscosity liquid formulation of inactivated poliovirus (IPV) which retains immunogenicity. The invention also includes avaccine comprising a dried solid or high viscosity liquid formulation ofIPV. A further aspect of the invention is a process for preservinginactivated polio virus (IPV) as a dried solid or high viscosity liquid.This process comprises preparing a sample by suspending or dissolvingIPV and a bacterial polysaccharide in a solution of a stabilising agentand subjecting the sample to temperature and pressure conditions whichresult in solvent being lost from the sample. Pressure and temperatureconditions are maintained or adjusted so that solvent is removed and thesample dries to form a solid or high viscosity liquid. Such formulationsmay be reconstituted prior to use or used directly.

IPV is well known as a component of vaccines, however, it is formulatedas a liquid, for example in Infanrix Penta®. The process offreeze-drying IPV has been associated with the loss of antigenicity sothat it is difficult to formulate an effective vaccine comprising adried form of IPV. Dried vaccine formulations are known, particularly inthe case of bacterial polysaccharides. The PRP polysaccharide ofHaemophilus influenzae b (Hib) is frequently formulated as a driedsolid, for example in Infanrix Hexa®(WO99/48525).

There are several reasons why a dried formulation of IPV would beadvantageous. Dried formulations have good storage properties and canincrease the shelf life of a vaccine containing IPV. The possibility ofdrying IPV also makes IPV a more flexible vaccine constituent andenables it to be formulated in new combination vaccines which were notpreviously possible. Some vaccines contain liquid and dried solidcomponents which are mixed just prior to administration (for exampleInfanrix Hexa®). Infanrix Hexa contains a dried Hib component which isreconstituted with DTPa-HepB-IPV just prior to use. By formulating IPVtogether with Hib as a dried solid, it would be possible to add furthercomponents to the liquid part of the vaccine, which might otherwise beincompatible with IPV.

Several techniques for drying vaccine components are known in the art.Traditionally, this has been accomplished using the process of freezedrying in which a solution of the substance is made and the sample isfrozen. During the primary drying phase, most of the water is removed bysublimation from ice under reduced pressure conditions and a porous‘cake’ is formed. This is usually followed by a secondary drying phasewhen the pressure and temperature are changed and water is evaporatedfrom the solid ‘cake’. The resulting lyophilised sample has improvedstability compared to a liquid formulation. However, the freeze dryingprocess is lengthy and can be the rate limiting step in a productionprocess.

Product variability is also a problem when many samples are being batchlyophilised in a large dryer unit. The conditions on the shelves of thefreeze dryer vary between different positions leading to sampleslyophilising at different rates under different conditions. For certainbiological materials such as live virus, there can be significant lossof activity during the freeze drying process (Pikal (1994) ACS Symposium567: 120-133). Many freeze dried substances are still unstable atambient temperature (Carpenter et al (1994) ACS Symposium 567; 134-147).

Damage caused by the process of freezing may be circumvented to somedegree by the use of cryoprotectants such as polyols. Furtherimprovements on the process of lyophilisation have also been made byavoiding freezing the sample during the process and removing water byboiling (WO96/40077; U.S. Pat. No. 6,306,345). This method involvespreparing a mixture of a glass-matrix forming material in a suitablesolvent together with the sample to be preserved, evaporating bulksolvent from the mixture to obtain a syrup, exposing the syrup to apressure and temperature sufficient to cause boiling of the syrup andremoving residual solvent.

A similar method was described in U.S. Pat. No. 5,766,520, in which theprocess involves partially removing the water to form a viscous fluidand further subjecting the syrup to vacuum to cause it to ‘boil’ andfurther drying at temperatures substantially lower than 100° C. Thismethod still suffers from some of the problems of conventionalfreeze-drying. When the process is carried out in a large freeze-dryer,samples will dry at different rates depending on their position on theshelf and this leads to different samples loosing different amount ofactivity during the drying process. This leads to a lack of consistencywithin a batch.

To date, no successful example of making a dried solid vaccineformulation of IPV that retains a high degree of antigenicity and/orimmunogenicity has been reported.

Accordingly, the present invention discloses an immunogenic compositioncomprising IPV and a stabilising agent, formulated as a driedcomposition or highly viscous liquid, which after reconstitution iscapable of generating an immune response against polio virus. Thepresence of a stabilising agent is crucial to the preservation ofantigens and polyols are shown to be effective. IPV is preferably driedin the presence of a bacterial polysaccharide which leads to retentionof a higher percentage of the original antigens in terms of antigenicityand/or immunogenicity. The present invention encompasses methods ofpreserving a composition comprising IPV, preferably in the presence of apolyol and a bacterial polysaccharide, wherein the antigenicity and/orimmunogenicity of IPV is retained. Lyophilisation of IPV in the presenceof polysaccharides leads to an improvement in antigen retention for IPVcompared to lyophilisation of IPV alone. In addition, the immunogenicityof Hib is also enhanced by being formulated together with IPV as a driedsolid or highly viscous liquid. In particular, when reconstitutedextemporaneously with liquid DTP vaccines (described below), theinventors have found that Hib titres are not as reduced by the aluminumhydroxide component of the DTP vaccine as would have been the casewithout the presence of IPV.

The method of drying used can also influence the antigenicity and/orimmunogenicity retention of IPV. A foam drying process for drying IPVwas more effective at retaining antigenicity of IPV than conventionalfreeze drying techniques. Surprisingly, the inclusion of a freezing stepin the foam drying process did not lead to loss of antigenicity butrather led to the development of a quick and effective preservationprocess. A further preferred method of the invention retains high levelsof IPV antigenicity and/or immunogenicity by drying the samplecontaining IPV without freezing or foam formation, resulting in theformation of a dried formulation, preferably a highly viscous liquidformulation.

The invention provides a dried formulation of IPV which will havebenefits of storage stability. The dried formulation can bereconstituted quickly and easily just prior to administration. Where thepreferred foam drying process is used, the foamed cake is particularlyeasily reconstituted due to the greater surface area of the cake.

Additional benefits of a dried solid or highly viscous liquidformulation of IPV and Hib include enhanced immunogenicity of the Hibcomponent. It is well known that in multi-component vaccines, otherparts of the vaccine formulation can lead to interference with Hibimmunogenicity (WO96/40242, WO97/00697). The inclusion of IPV in a driedformulation with Hib can reduce this problem, especially if the driedIPV-Hib composition is mixed with diphtheria, tetanus and pertussiscomponents prior to administration.

Although lyophilisation of IPV in the presence of a bacterialpolysaccharide is possible using a conventional freeze drying approach,it is preferred to use a foam drying technique or a gentle dryingprocess which does not involve freezing or foam formation. Theseprocesses result in even greater antigenicity and/or immunogenicityretention in IPV and the resultant cake is also easier and quicker toreconstitute. The processes also have advantages in being quicker andmore energy efficient than standard freeze-drying techniques. Since thelyophilisation step is often the rate limiting step in vaccineproduction, the use of the preferred processes would result in higherlevels of vaccine production without additional investment in plant. Theintroduction of a freezing step into the preferred foam drying processalso leads to improved batch reproducibility.

DESCRIPTION OF FIGURES

FIG. 1—Photographs of vials containing the preservation sample atdifferent stages of the foam drying process.

A—Shows the appearance of the preservation samples as inserted into thefreeze drying as a liquid formulation.

B—Shows the appearance of the preservation samples as the pressure isreduced to 1.5 mbars. The samples begin to freeze at slightly differentrates due to differing conditions in each vial.

C—Shows the appearance of the preservation samples at 0.1 mbars, whereall samples have become completely frozen.

D—Shows the appearance of the preservation samples as the pressure isincreased to 0.8-3.5 mbars. A foamed glass is formed as the preservationsample foams and solvent evaporates.

FIG. 2—Photograph of the highly viscous liquid in inverted vials.

DETAILED DESCRIPTION

Immunogenic Compositions of the Invention

The invention includes immunogenic compositions, formulated as a driedsolid or a highly viscous liquid comprising IPV and a stabilising agent,in which the antigenicity and/or immunogenicity of IPV is retainedfollowing reconstitution. The dried solid or highly viscous liquidformulation of IPV is capable of generating an immune response,preferably a protective immune response, against polio virus, preferablyafter reconstitution and inoculation.

IPV is defined as inactivated polio virus (preferably comprising types1, 2 and 3 as is standard in the vaccine art, most preferably the Salkpolio vaccine). A vaccine dose of IPV contains 20-80, preferably 40 or80 D-antigen units of type 1 Mahoney), 4-16, preferably 8 or 16D-antigen units of type 2 (MEF-1) and 20-64, preferably 32 or 64D-antigen units of type 3 (Saukett).

When dried by a method of the invention, preferably the antigenicity of1, 2, or all 3 of types 1, 2 and 3 of polio virus are retained; morepreferably the antigenicity of type 1; type 2; type 3; type 1 and type2; type 1 and type 3; type 2 and type 3; or type 1, type 2 and type 3 isretained at a level of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98%of the antigenicity of a reference sample which has not been subjectedto the drying process. This can be measured, following reconstitution ofthe dried solid or highly viscous liquid in an aqueous solution, by anysuitable method including by ELISA using polyclonal and/or monoclonalantibodies against polio virus type 1, 2 and/or 3.

When dried by a method of the invention, preferably the immunogenicityof 1, 2, or all 3 of types 1, 2 and 3 of polio virus are retained; morepreferably the immunogenicity of type 1; type 2; type 3; type 1 and type2; type 1 and type 3; type 2 and type 3; or type 1, type 2 and type 3 isretained at a level of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98%of the immunogencity of a reference sample which has not been subjectedto the drying process. This can be measured, following reconstitution ofthe dried solid or highly viscous liquid in an aqueous solution, by anysuitable method. In a preferred method, the dried formulation isreconstituted in an aqueous solution and is inoculated into an animal,preferably a rat. After a suitable period of time, antisera arecollected from the inoculated animals and seroconversion is tested.Preferably, a relative potency of at least 0.4, 0.5, 0.6, 0.7, 0.8 or0.9 is achieved, compared to an undried reference sample.

A dried solid composition is a formulation which has had solvent removedby a process of lyophilisation, sublimation, evaporation or desiccationso that less than or equal to 15%, 12%, 10%, 7%, 5%, 4%, preferably 3%,2% or most preferably 1%. solvent remains. The term ‘dried solid’comprises glasses, rubbers or crystalline solids with a solidappearance. Any of the methods described above can be used to make sucha dried solid. Solvent is removed by sublimation, boiling orevaporation, preferably by evaporation.

A highly viscous liquid is defined as a material with a solvent contentless than or equal to 15, 12, 10, preferably 8, 5, 4, 3, 2 or 1%. Thehighly viscous liquid has a sufficiently low solvent content such thatthe active agent is preserved in a stable state for at least 3,6,9,12 or24 months at 4° C., allowing the active agent to retain at least 40, 50,60, preferably 70, 80, 90, 95% of its antigenicity and/or immunogencityover this period. The highly viscous liquid has not been exposed to theformations of bubbles that is involved in foam formation. Preferably,the highly viscous liquid has a solid appearance but is a glass and isable to flow very slowly over a period of days, preferably weeks, morepreferably months.

Immunogenic compositions of the invention are formulated as a driedsolid or highly viscous liquid comprising IPV and a stabilising agentand preferably a bacterial polysaccharide. The stabilising agent is anyof the compositions described below. The bacterial polysaccharidecomprises capsular polysaccharides derived from any bacterium,preferably one or more of Neisseria meningitidis, Haemophilus influenzaeb, Streptococcus pneumoniae, Group A Streptococci, Group B Streptococci,Staphylococcus aureus or Staphylococcus epidermidis.

Preferably the PRP capsular polysaccharide of Haemophilus influenzae bis present as a dried solid or highly viscous liquid. In a furtherpreferred embodiment, the immunogenic composition comprises dried solidor highly viscous liquid formulations of capsular polysaccharidesderived from one or more of serogroups A, C, W-135 and Y of Neisseriameningitidis (meningococcal polysaccharides). A further preferredembodiment comprises dried solid or highly viscous liquid formulationsof capsular polysaccharides derived from Streptococcus pneumoniae. Thepneumococcal capsular polysaccharide antigens are preferably selectedfrom serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B,17F, 18C, 19A, 19F, 20, 22F, 23F and 33F (most preferably from serotypes1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F). A further preferredembodiment contains the Type 5, Type 8 or 336 capsular polysaccharidesof Staphylococcus aureus. A further preferred embodiment contains theType I, Type II or Type m capsular polysaccharides of Staphylococcusepidermidis. A further preferred embodiment contains the Type Ia, TypeIc, Type II or Type III capsular polysaccharides of Group Bstreptocoocus. A further preferred embodiment contains the capsularpolysaccharides of Group A streptococcus, preferably further comprisingat least one M protein and more preferably multiple types of M protein.

In one embodiment of the invention, the bacterial polysaccharides arefull length, being purified native polysaccharides. In an alternativeembodiment of the invention, the polysaccharides are sized between 2 and20 times, preferably 2-5 times, 5-10 times, 10-15 times or 15-20 times,so that the polysaccharides are smaller in size for greatermanageability. Oligosaccharides are used in a preferred embodiment.Oligosaccharides typically contain between 2 and 20 repeat units.

The invention further includes immunogenic compositions comprising morethan one bacterial polysaccharide and IPV as a dried solid or highlyviscous liquid. Preferably, IPV is combined with one or more of Hib(Haemophilus influenzae type b) PRP polysaccharide and/or meningococcalA, C, W and/or Y polysaccharides and/or pneumococcal polysaccharides.Most preferably the active agents comprise, IPV and Hib; IPV and MenC;IPV and Hib and MenC; IPV and MenA and C; IPV and Hib and Men A and C;IPV and Hib and Men A and C and Y; or IPV and Hib and Men C and Y.

The above particularised active agents may also comprise one or morepneumococcal capsular polysaccharides as described below.

In the above compositions where polysaccharides are used,oligosaccharides may also be employed (as defined above).

Although these compositions may be adjuvanted (as described below), theyare preferably unadjuvanted or preferably do not comprise aluminiumsalts.

Preferably the polysaccharides or oligosaccharides are conjugated to apeptide or carrier protein comprising T-helper epitopes (as describedbelow).

Capsular polysaccharides present in immunogenic compositions of theinvention are unconjugated or conjugated to a carrier protein such astetanus toxoid, tetanus toxoid fragment C, diphtheria toxoid, CRM197,pneumolysin, Protein D (U.S. Pat. No. 6,342,224). Tetanus toxin,diphtheria toxin and pneumolysin are detoxified either by geneticmutation and/or preferably by chemical treatment. A preferred embodimentof the invention has Hib conjugated to tetanus toxoid.

Where more than one conjugated polysaccharide is present in theimmunogenic composition of the invention, the polysaccharides areconjugated to the same carrier protein or to different carrier proteins.Preferred embodiments of the invention contain meningococcalpolysaccharides conjugated to a carrier protein. Where conjugated Hiband meningococcal polysaccharides are present, they are conjugated tothe same carrier protein or to different carrier proteins.

The polysaccharide conjugate may be prepared by any known couplingtechnique. In a preferred coupling technique, the polysaccharide iscoupled via a thioether linkage. This conjugation method relies onactivation of the polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may thus be coupled directly or via a spacer group to anamino group on the carrier protein. Preferably, the cyanate ester iscoupled with hexane diamine and the amino-derivatised polysaccharide isconjugated to the carrier protein using heteroligation chemistryinvolving the formation of the thioether linkage. Such conjugates aredescribed in PCT published application WO93/15760 Uniformed ServicesUniversity.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508.

A further method involves the coupling of a cyanogen bromide activatedpolysaccharide derivatised with adipic acid hydrazide (ADH) to theprotein carrier by Carbodiimide condensation (Chu C. et al Infect.Immunity, 1983 245 256).

Polysaccharides which are incorporated as part of the immunogeniccomposition of the invention may be unabsorbed or absorbed onto anadjuvant, preferably an aluminium salt (aluminium phosphate or aluminiumhydroxide), most preferably aluminium phosphate.

Immunogenic compositions of the invention comprise a stabilising agentwhich can help to prevent damage during the desiccation process. Any ofthe stabilising agent described below, including glass forming polyolscan be incorporated into the immunogenic composition, whether as a driedsolid, a foamed glass or a highly viscous liquid composition using theprocesses of the invention. Preferred stabilising agents includesucrose, sorbitol, lactose and trehalose.

The preferred combinations, dried by the processes of the invention maybe combined with other antigens in a combination vaccine which aredesiccated or liquid formulations which are used to reconstitute thedried components.

Additional Components

Dried solid or highly viscous liquid formulations of the inventionincorporating IPV and a stabilising agent may additionally be formulatedwith further vaccine components. A preferred vaccine contains a driedsolid or highly viscous liquid formulation of IPV and a bacterialpolysaccharide which may be mixed with a liquid formulation comprisingadditional vaccine components. After reconstitution of the solidcomponents with the liquid components, the complete vaccine isadministered by injection.

The additional components include capsular polysaccharides derived fromone or more of Neisseria meningitidis, Streptococcus pneumoniae, Group AStreptococci, Group B Streptococci, Staphylococcus aureus orStaphylococcus epidermidis. In a preferred embodiment, the immunogeniccomposition comprises capsular polysaccharides derived from one or moreof serogroups A, C, W-135 and Y of Neisseria meningitidis. A furtherpreferred embodiment comprises capsular polysaccharides derived fromStreptococcus pneumoniae. The pneumococcal capsular polysaccharideantigens are preferably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F,8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and33F (most preferably from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19Fand 23F). A further preferred embodiment contains the Type 5, Type 8 or336 capsular polysaccharides of Staphylococcus aureus. A furtherpreferred embodiment contains the Type I, Type II or Type III capsularpolysaccharides of Staphylococcus epidermidis. A further preferredembodiment contains the Type Ia, Type Ic, Type II or Type III capsularpolysaccharides of Group B streptocoocus. A further preferred embodimentwould contain the capsular polysaccharides of Group A streptococcus,preferably further comprising at least one M protein and more preferablymultiple types of M protein.

The immunogenic composition of the invention may be formulated withprotein antigens. Preferred pneumococcal proteins antigens are thosepneumococcal proteins which are exposed on the outer surface of thepneumococcus (capable of being recognised by a host's immune systemduring at least part of the life cycle of the pneumococcus), or areproteins which are secreted or released by the pneumococcus. Mostpreferably, the protein is a toxin, adhesin, 2-component signaltranducer, or lipoprotein of Streptococcus pneumoniae, or fragmentsthereof. Particularly preferred proteins include, but are not limitedto: pneumolysin (preferably detoxified by chemical treatment ormutation) [Mitchell et al. Nucleic Acids Res. 1990 Jul. 11; 18(13): 4010“Comparison of pneumolysin genes and proteins from Streptococcuspneumoniae types 1 and 2.”, Mitchell et al. Biochim Biophys Acta 1989Jan. 23; 1007(1): 67-72 “Expression of the pneumolysin gene inEscherichia coli: rapid purification and biological properties.”, WO96/05859 (A. Cyanamid), WO 90/06951 (Paton et al), WO 99/03884 (NAVA)];PspA and transmembrane deletion variants thereof (U.S. Pat. No.5,804,193—Briles et al.); PspC and transmembrane deletion variantsthereof (WO 97/09994—Briles et al); PsaA and transmembrane deletionvariants thereof (Berry & Paton, Infect Immun 1996 Dec.; 64(12):5255-62“Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesinessential for virulence of Streptococcus pneumoniae”); pneumococcalcholine binding proteins and transmembrane deletion variants thereof;CbpA and transmembrane deletion variants thereof (WO 97/41151; WO99/51266); Glyceraldehyde-3-phosphate-dehydrogenase (Infect. Immun. 199664:3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beato et al. FEMS MicrobiolLett 1998, 164:207-14); M like protein, (EP 0837130) and adhesin 18627,(EP 0834568). Further preferred pneumococcal protein antigens are thosedisclosed in WO 98/18931, particularly those selected in WO 98/18930 andPCT/US99/30390.

Preferred Neisserial proteins to be formulated with the immunogeniccomposition of the invention include ThpA (WO93/06861; EP586266;WO92/03467; U.S. Pat. No. 5,912,336), TbpB (WO93/06861; EP586266), Hsf(WO99/31132), NspA (WO96/29412), Hap (PCT/EP99/02766), PorA, PorB, OMP85(also known as D15) (WO00/23595), PilQ (PCT/EP99/03603), PldA(PCT/EP99/06718), FrpB (WO96/31618 see SEQ ID NO:38), FrpA or FrpC or aconserved portion in common to both of at least 30, 50, 100, 500, 750amino acids (WO92/01460), LbpA and/or LbpB (PCT/EP98/05117; Schryvers etal Med. Microbiol. 1999 32: 1117), FhaB (WO98/02547), HasR(PCT/EP99/05989), lipo02 (PCT/EP99/08315), MltA (WO99/57280) and ctrA(PCT/EP00/00135). Neisserial protein may be added as purified proteinsor as part of an outer membrane vesicle preparation.

The immunogenic composition is preferably formulated with antigensproviding protection against one or more of Diphtheria, tetanus andBordetella pertussis infections. The pertussis component may be killedwhole cell B. pertussis (Pw) or is preferably a cellular pertussis (Pa)which contains at least one antigen (preferably two or all three) fromPT, FHA and 69 kDa pertactin certain other a cellular vaccines alsocontain agglutinates, such as Film 2 and Film 3 and these vaccines arealso contemplated for use in the invention. Typically, the antigensproviding protection against Diphtheria and Tetanus are Diphtheriatoxoid and tetanus toxoid. The toxoids are chemically inactivatedtoxins, for example following treatment with formaldehyde, or toxinsinactivated by the introduction of one or more point mutations.

Alternatively the immunogenic composition of the invention may beprovided as a kit with the dried solid, foamed glass or highly viscousliquid in one container and liquid DTPa or DTPw in another container.Such kits can for example, comprise a dual chamber syringe with thedried and liquid components contained in the same syringe but indifferent chambers. The dried component is then reconstituted with theliquid vaccine immediately prior to injection as a single vaccine. Thusfor example, the dried solid, foamed glass or highly viscous liquid ofthe invention is reconstituted with the liquid DTPa or DTPw vaccine(preferably extemporaneously) and administered as a single vaccine. TheDTPa or DTPw vaccine typically is adjuvanted at least in part with analuminium salt, such as aluminium phosphate and/or aluminium hydroxide(for instance Infanrix® and Tritanrix® vaccines of GlaxoSmithKlineBiologicals s.a.).

The immunogenic composition is optionally formulated with one or moreantigens that can protect a host against non-typeable Haemophilusinfluenzae, RSV and/or one or more antigens that can protect a hostagainst influenza virus. Preferred non-typeable H. influenzae proteinantigens include Fimbrin protein (U.S. Pat. No. 5,766,608) and fusionscomprising peptides therefrom (eg LB1 Fusion) (U.S. Pat. No.5,843,464—Ohio State Research Foundation), OMP26, P6, protein D, ThpA,TbpB, Hia, Hmw1, Hmw2, Hap, and D15.

Preferred influenza virus antigens include whole, live or inactivatedvirus, split influenza virus, grown in eggs or MDCK cells, or Vero cellsor whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10,915-920) or purified or recombinant proteins thereof, such as HA, NP,NA, or M proteins, or combinations thereof.

Preferred RSV (Respiratory Syncytial Virus) antigens include the Fglycoprotein, the G glycoprotein, the HN protein, the M protein orderivatives thereof.

Combination vaccines comprising DTP-Hib are known in the art. Howeverthere are problems associated with certain formulations which involvesimple mixing of Hib with other antigens. Unless carefully formulation,the antibody titres raised against the Hib component can be lower thanthose elicited by the same dose of Hib inoculated separately, due tointerference with other components of the vaccine. Although this problemis well known in the art and has been addressed in various ways, theimmunogenic compositions of the invention in which Hib and IPV areformulated together as a dried solid or highly viscous liquid providesan alternative solution to this problem.

The immunogenic compositions of the invention may form part of a vaccinekit in which IPV and Hib are present in one component of the kit andfurther components, as described above, are present in a secondcomponent, for example, a dual chamber syringe as described herein. Thetwo components are mixed together just before administration of thevaccine. In such formulations, the component comprising IPV and Hib ispreferably a dried solid, foamed glass or highly viscous liquid,although it is optionally formulated as a liquid. This formulationresults in antibody titres against the Hib component being clinicallyacceptable to provide protection against the Haemophilus influenzae bpathogen. Typically, the antibody titre in the combination vaccine areat least 85%, 90%, preferably about 100% or more of those elicited bythe same dose of Hib in a monovalent Hib vaccine.

Vaccines of the Invention

The immunogenic compositions of the invention described above arepreferably formulated as a vaccine. Preferably, the vaccine contains anamount of an adjuvant sufficient to enhance the immune response to theimmunogen. Suitable adjuvants include, but are not limited to, aluminiumsalts such as aluminium hydroxide and aluminium phosphate, squalenemixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacteriumcell wall preparations, monophosphoryl lipid A, mycolic acidderivatives, non-ionic block copolymer surfactants, Quil A, choleratoxin B subunit, polphosphazene and derivatives, and immunostimulatingcomplexes (ISCOMs) such as those described by Takahashi et al. (1990)Nature 344:873-875. For veterinary use and for production of antibodiesin animals, mitogenic components of Freund's adjuvant can be used.

The vaccine formulations of the invention are preferably reconstitutedprior to use. Reconstitution involves the mixing of a liquid componentof the vaccine with the dried solid, foamed glass or highly viscousliquid formulation of the invention. The invention also encompasses acontainer with a water repellent internal surface containing theimmunogenic composition or vaccine of the invention. The use of such acontainer is advantageous because it leads to the dried compositionsitting at the bottom of the tube in a form in which it is more easy toreconstitute.

It is advantageous to incorporate a coloured dye into the preservationsample in order to allow easier visualisation of the dried compositionof the invention. This is particularly important during reconstitutionto ensure that the dried solid or highly viscous liquid is thoroughlyreconstituted prior to use. Preferably, the coloured dye maintains itscolour at a neutral pH and is compatible with injection into a patient.Most preferably the coloured dye is phenol red.

As with all immunogenic compositions or vaccines, the immunologicallyeffective amounts of the immunogens must be determined empirically.Factors to be considered include the immunogenicity, whether or not theimmunogen will be complexed with or covalently attached to an adjuvantor carrier protein or other carrier, route of administrations and thenumber of immunising dosages to be adminstered. Such factors are knownin the vaccine art and it is well within the skill of immunologists tomake such determinations without undue experimentation.

The substance can be present in varying concentrations in theimmunogenic composition of the invention. Typically, the minimumconcentration of the substance is an amount necessary to achieve itsintended use, while the maximum concentration is the maximum amount thatwill remain in solution or homogeneously suspended within the initialmixture. For instance, the minimum amount of a therapeutic agent ispreferably one which will provide a single therapeutically effectivedosage. Super-saturated solutions can also be used if a foamed glass isformed prior to crystallisation. For bioactive substances, the minimumconcentration is an amount necessary for bioactivity upon reconstitutionand the maximum concentration is at the point at which a homogeneoussuspension cannot be maintained. In the case of single-dosed units, theamount is that of a single therapeutic application Generally, it isexpected that each dose will comprise 1-100 ug of protein antigen,preferably 5-50 ug and most preferably 5-25 ug. Preferred doses ofbacterial polysaccharides are 10-20 ug, 10-5 ug, 5-2.5 ug or 2.5-1 ug.The preferred amount of the substance varies from substance to substancebut is easily determinable by one of skill in the art.

Methods of the Invention

The methods of the invention are for preserving a composition comprisingIPV and a stabilising agent, resulting in a composition in which theantigenicity of IPV is retained. Preferably, a bacterial polysaccharideis incorporated in the sample to be dried.

In one embodiment, the method of the invention involves drying IPV andcomprises the steps of:

-   -   preparing a preservation sample by suspending or dissolving IPV        in a solution of a stabilising agent; preferably a bacterial        polysaccharide and/or a glass forming polyol are present in the        preservation sample;    -   subjecting the preservation sample to such temperature and        pressure conditions that solvent is lost from the preservation        sample; and    -   removing solvent until the preservation sample dries to form a        solid or highly viscous liquid in which the antigenicity and/or        immunogenicity of IPV is retained.

In a preferred embodiment, the preservation sample is inserted into acontainer with a water repellent interior prior to drying.

A further method of the invention involves foam drying, comprising thesteps of:

-   -   preparing a preservation sample by suspending or dissolving IPV        in a solution of a stabilising agent; preferably a bacterial        polysaccharide and/or a glass forming polyol are present in the        preservation sample;    -   subjecting the preservation sample to such temperature and        pressure conditions that the preservation sample forms a foam;        and    -   removing solvent until the foam dries to form a solid in which        the antigenicity and/or immunogenicity of IPV is retained.

A preferred foam drying method of the invention uses a container with awater repellent interior surface and contains the steps of:

-   -   preparing a preservation sample by suspending or dissolving IPV        and preferably a bacterial polysaccharide in a solution of a        stabilising agent;    -   inserting the preservation sample into a container with a water        repellent interior surface;    -   subjecting the container containing the preservation sample to        such temperature and pressure conditions so that the        preservation sample forms a foam;    -   removing solvent until the foam dries to form a solid in which        the antigenicity and/or immunogenicity of IPV is retained.

The foam drying methods of the invention described above optionallycomprise a freezing step. The preservation sample may be wholly orpartially frozen. Therefore some methods of the invention comprise thesteps of:

-   -   preparing an at least partially frozen preservation sample by        suspending or dissolving IPV and preferably a bacterial        polysaccharide in a solution of a stabilising agent and freezing        the mixture;    -   subjecting the at least partially frozen preservation sample to        such temperature and pressure conditions that the preservation        sample forms a foam; and    -   removing solvent until the foam dries to form a solid in which        the antigenicity and/or immunogenicity of IPV is retained.

The freezing step of the above method is preferably by the process ofquench freezing in which reduction of pressure is the cause of freezingby evaporation. This causes rapid freezing of the sample which leads toless antigen loss. Therefore a process of the invention includes thesteps of:

-   -   preparing a preservation sample by suspending or dissolving IPV        and preferably a bacterial polysaccharide in a solution of a        stabilising agent;    -   subjecting the preservation sample to reduced pressure such that        the preservation sample becomes at least partially frozen;    -   subjecting the at least partially frozen preservation sample to        such temperature and pressure conditions that the preservation        sample forms a foam; and    -   removing solvent until the foam dries to form a solid in which        the antigenicity and/or immunogenicity of IPV is retained.

A further preferred method of the invention is used for preserving IPVand comprises the steps of:

-   -   preparing a preservation sample by suspending or dissolving IPV        in a solution of a stabilising agent;    -   subjecting the preservation sample to such temperature and        pressure conditions that the preservation sample looses solvent        by evaporation, without bubbling to form a foam and preferably        without freezing;    -   removing solvent until the sample dries to form a highly viscous        liquid in which and antigencity and/or immunogenicity of IPV is        retained.

The methods of the invention produce a formulation of IPV that is ableto withstand extended storage during which the antigenicity and/orimmunogenicity of IPV is maintained. Preferably the IPV retains at least40, 50, 60, 70, preferably 80, 90, 95% of its original antigenicityand/or immunogenicity over a period of at least 3, 6, 9, 12, 24 monthsstorage at 4° C. Antigenicity and immunogenicity are measured afterreconstitution of IPV in a suitable aqueous solution, and using asuitable method, for instance those described above.

The method of drying without freezing or foam formation is particularlyapplicable for use where the active agents to be dried are prone to lossof activity and/or antigenicity during the drying process due toexposure to freezing or the bubbling associated with foam formation. Itis also particularly applicable for use where a lower concentration ofthe glass forming polyol is advantageous and/or where a shorter dryingprocess is preferred.

Stabilising Agent

The stabilising agent to be used in the methods of the invention willpreferably comprise glass forming polyols. Suitable materials include,but are not limited to, all polyols, including carbohydrate andnon-carbohydrate polyols. Preferably the stabilising polyol enables theactive agent to be stored without substantial loss of activity bydenaturation, aggregation or other means. Particularly suitablematerials include sugars, sugar alcohols and carbohydrate derivatives.Preferably, the glass forming polyol is a carbohydrate or derivativesthereof, including glucose, maltulose, iso-maltulose, lactulose,sucrose, maltose, lactose, iso-maltose, maltitol, lactitol, palatinit,trehalose, raffinose, stachyose, melezitose or dextran, most preferablytrehalose, sucrose, sorbitol, raffinose, mannitol, lactose, lactitol orpalatinit.

Bacterial polysaccharides act as a stabilising agent and preferredembodiments of the invention incorporate bacterial polysaccharides as aconstitutent of the stabilising agent. The bacterial polysaccharideplays a dual role of stabilising agent and immunogen in this embodiment.

Carbohydrates include, but are not limited to, monosaccharides,disaccharides, trisaccharides, oligosaccharides and their correspondingsugar alcohols, polyhydroxyl compounds such as carbohydrate derivativesand chemically modified carbohydrates, hydroxyethyl starch and sugarcopolymers. Both natural and synthetic carbohydrates are suitable foruse. Synthetic carbohydrates include, but are not limited to, thosewhich have the glycosidic bond replaced by a thiol or carbon bond. BothD and L forms of the carbohydrates may be used. The carbohydrate may benon-reducing or reducing. Where a reducing carbohydrate is used, theaddition of inhibitors of the Maillard reaction is preferred.

Reducing carbohydrates suitable for use in the invention are those knownin the art and include, but are not limited to, glucose, maltose,lactose, fructose, galactoase, mannose, maltulose and lactulose.Non-reducing carbohydrates include, but are not limited to, non-reducingglycosides of polyhydroxyl compounds selected from sugar alcohols andother straight chain polyalcohols. Other useful carbohydrates includeraffinose, stachyose, melezitose, dextran, sucrose, cellibiose,mannobiose and sugar alcohols. The sugar alcohol glycosides arepreferably monoglycosides, in particular the compounds obtained byreduction of disaccharides such as lactose, maltose, lactulose andmaltulose.

Particularly preferred carbohydrates are trehalose, sucrose, sorbitol,maltitol, lactitol, palatinit and glucopyranosyl-1→6-mannitol.

Amino acids can act as stabilising agents and can be used by themselvesand preferably in combination with a polyol. Preferred amino acidsinclude glycine, alanine, arginine, lysine and glutamine although anyamino acid, or a combination of amino acids, peptide, hydrolysedproteins or protein such as serum albumin can act as a stabilisingagent.

Preferably, the preservation sample will contain a component capable ofinhibiting crystal formation in the dried solid or highly viscous liquidof the invention. Salts and other molecules including amino acids andphenol red inhibit crystal formation.

The concentration of the stabilising agent used in the process of theinvention may be between 1% and 50% weight/volume, preferably 1-5%,5-10%, 5-10%, 15-20%, 20-25% or 25-50%, most preferably less than 25%(w/v). The amounts of stabilising agent required is proportional to theamount of salts present. Therefore, although levels of stabilising agentbetween 3% and 10% are preferred, higher concentrations of 10% to 25%(w/v) may be required to dry samples with a high salt content.

Container

Different mixtures and various container shapes and sizes can beprocessed simultaneously. Ideally, the container size used is sufficientto contain the initial mixture and accommodate the volume of the driedformulation formed thereof. Typically, this is determined by the mass ofthe glass forming material, the surface area of the container and thetemperature and pressure conditions, which determine whether foamingoccurs. The mass of glass forming material must be sufficient to giveviscous syrup, optionally to be foamed which translates practically as aminimal mass per unit area of container surface. This ratio varies frommixture to mixture and container used, but is easily determinedempirically by one skilled in the art by following the procedures setforth herein. Any such containers can be used including Wheaton mouldedand tube-cut vials.

The processes of the invention preferably use containers with a waterrepellent interior surface. This is achieved through coating theinterior surface with a hydrophobic composition, for instance bysiliconisation. Siliconisation is achieved by processes that are wellknown to those skilled in the art. In one method, the container issiliconised by rising the interior of the container with an emulsion ofsilicone, followed by processing through an oven at high temperature,typically 350° C. Alternatively, the water repellent interior surface isachieved by the container being made of a water repellent composition.

The water repellent interior surface of the container makes foamformation more likely to occur and more reproducible. This allows lowerpolyol concentrations to be used in the preservation sample which inturn decreases the length of time necessary to dry the sample, reducesthe effect of Maillard reactions or other interactions with the polyolharming the active agent. Where the preservation samples comprises avaccine, the resultant foamed glass is reconstituted quickly and easilydue to the lower amount of polyol present and the resultant vaccinesolution is less viscous, allowing easier administration. The waterrepellent interior surface allows easier reconstitution of the driedsolid or highly viscous liquid since it encourages the sample to remainas at the bottom of the container so that it is easier to reconstituteeffectively.

Although singular forms may be used herein, more than one stabilisingagent, more than one additive, and more than one substance may bepresent. Effective amounts of these components are easily determined byone skilled in the art.

Solvent

The preservation sample is made by dissolving/suspending IPV and astabilising agent in water to make an aqueous solution. Preferably,water is present in the preservation sample at a level of 5 to 98% byvolume, more preferably 80-98% by volume, most preferably 85-98% byvolume.

The volume of solvent can vary and will depend upon the stabilisingagent and the substance to be incorporated as well as any additives. Theminimum volume required is an amount necessary to solubilise the variouscomponents. However, homogeneously dispersed suspensions of thesubstance(s) can also be used. Suitable amounts of the components inspecific embodiments are easily determinable by those skilled in the artin light of the examples provided herein.

Various additives can be put into the preservation sample. Typically,the additives enhance foam formation and/or the drying process and/orcontribute to the solubilization of the substance. Alternatively, theadditives contribute to the stability of the substance incorporatedwithin the solid. One or more additives may be present.

As an example, addition of volatile/effervescent salts allows largerinitial volumes and results in higher surface area within the foamedglass, thus effecting superior foam formation and more rapid drying. Asused herein, volatile salts are salts which volatilise under theconditions used to produce a foamed glass. Examples of suitable volatilesalts include, but are not limited to, ammonium acetate, ammoniumbicarbonate and ammonium carbonate. Salts that decompose to give gaseousproducts also effect enhanced foam formation and more rapid drying.Examples of such salts are sodium bicarbonate and sodium metabisulphite.Preferably, the volatile salts are present in an amount of from about0.01 to 5 M. Concentrations of up to 5 M are suitable for use herein.The resultant foamed glass has uniform foam conformation and issignificantly drier compared to foamed glass in whichvolatile/effervescent salts are not used.

Another suitable additive is a foam stabilising agent, which can be usedin combination with either the volatile or decomposing salt. This mayeither be a surface active component such as an amphipathic molecule(i.e. such as phospholipids and surfactants) or an agent to increase theviscosity of the foaming syrup, such as a thickening agent such as guargum and their derivatives.

Another additive is an inhibitor of the Maillard reaction. Preferably,if the substance and/or glass matrix-forming material contains carbonyland amino, imino or guanidino groups, the compositions further containat least one physiologically acceptable inhibitor of the Maillardreaction in an amount effective to substantially prevent condensation ofamino groups and reactive carbonyl groups in the composition. Theinhibitor of the Maillard reaction can be any known in the art. Theinhibitor is present in an amount sufficient to prevent, orsubstantially prevent, condensation of amino groups and reactivecarbonyl groups. Typically, the amino groups are present on thesubstance and the carbonyl groups are present on the glass matrixforming material, or the converse. However, the amino acids and carbonylgroups may be intramolecular within either the substance or thecarbohydrate.

Various classes of compounds are known to exhibit an inhibiting effecton the Maillard reaction and hence to be of use in the compositionsdescribed herein. These compounds are generally either competitive ornon-competitive inhibitors of the Maillard reaction. Competitiveinhibitors include, but are not limited to, amino acid residues (both Dand L), combinations of amino acid residues and peptides. Particularlypreferred are lysine, arginine, histidine and tryptophan. Lysine andarginine are the most effective. There are many known non-competitiveinhibitors. These include, but are not limited to, aminoguanidine andderivatives and amphotericin B. EP-A-0 433 679 also describes suitableMaillard inhibitors which include 4-hydroxy-5,8-dioxoquinolinederivatives.

Active Agents

The methods of the invention are used to preserve inactivated poliovirus (IPV—preferably comprising types 1, 2 and 3 as is standard in thevaccine art, most preferably the Salk polio vaccine). IPV contains20-80, preferably 40 or 8-D-antigen units of type 1 (Mahoney), 4-20,preferably 8 or 16 D-antigen units of type 2 (MEF-1) and 20-64,preferably 32 or 64 D-antigen units of type 3 (Saukett). The IPV vaccineformulation is suitable for injection after reconstitution in an aqueoussolution which preferably contains additional vaccine components.

The bacterial polysaccharide incorporated by the process of theinvention are for example capsular polysaccharides derived from one ormore of Neisseria meningitidis, Haemophilus influenzae b, Streptococcuspneumoniae, Group A Streptococci, Group B Streptococci, Staphylococcusaureus or Staphylococcus epidermidis, preferably the PRP capsularpolysaccharides of Haemophilus influenzae. Preferred capsularpolysaccharides also include those derived from one or more ofserogroups A, C, W-135 and Y of Neisseria meningitidis. Furtherpreferred capsular polysaccharides are derived from Streptococcuspneumoniae. The pneumococcal capsular polysaccharide antigens arepreferably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V,10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F (mostpreferably from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F).A further preferred embodiment contains the Type 5, Type 8 or 336capsular polysaccharides of Staphylococcus aureus. Further preferredpolysaccharides include the Type I, Type II or Type III capsularpolysaccharides of Staphylococcus epidermidis, the Type Ia, Type Ic,Type II or Type III capsular polysaccharides of Group B streptocoocus.Further preferred polysaccharides include the capsular polysaccharidesof Group A streptococcus, preferably further comprising at least one Mprotein and more preferably multiple types of M protein.

Preferred combinations of active agents to be preserved using theprocesses of the invention comprise IPV. Preferably, IPV is combinedwith bacterial polysaccharides comprising one or more of Hib PRPpolysaccharide and/or meningococcal A, C, W and/or Y polysaccharidesand/or pneumococcal polysaccharides. Preferred combinations include IPVand Hib; IPV and MenC; IPV and MenA and C; IPV and Hib and Men C or IPV,Hib, Men A and C. Each bacterial polysaccharides may be present in dosesof 1-5 μg, 5-10 μg, 10-20 μg or 20-40 μg.

Bacterial polysaccharides are unconjugated or conjugated to a carrierprotein such as tetanus toxoid, tetanus toxoid fragment C, diphtheriatoxoid, CRM197, pneumolysin or Protein D (U.S. Pat. No. 6,342,224).

The polysaccharide conjugate are prepared by any known couplingtechnique. A preferred conjugation method relies on activation of thepolysaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester. The activated polysaccharide is coupleddirectly or via a spacer group to an amino group on the carrier protein.Preferably, the cyanate ester is coupled with hexane diamine and theamino-derivatised polysaccharide is conjugated to the carrier proteinusing heteroligation chemistry involving the formation of the thioetherlinkage. Such conjugates are described in PCT published applicationWO93/15760 Uniformed Services University.

The conjugates are optionally prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508.

A further method involves the coupling of a cyanogen bromide activatedpolysaccharide derivatised with adipic acid hydrazide (ADH) to theprotein carrier by Carbodiimide condensation (Chu C. et al Infect.Immunity, 1983 245 256).

Drying Processes

In one embodiment the process of the invention involves drying IPV inthe presence of a stabilising agent, preferably in the presence of abacterial polysaccharide. In this process, the preservation sample issubjected to reduced temperature and pressure conditions. Thetemperature is reduced to less than 20° C. or 0° C., preferably lessthan −10° C. or −20° C., more preferably −40° C. or −60° C. The pressureis reduced to less than 1 mbar, preferably a pressure of, or less than0.5, 0.1, more preferably 0.05 or 0.01 mbar. The reduced temperature andpressure conditions are maintained for at least 10, 12, 16, 20,preferably 24, 36, more preferably 48 or 72 hours. Solvent is removeduntil the preservation sample dries to form a solid.

Throughout this application, solid includes glasses, rubbers andcrystals which form as the sample dries. Such solids preferably retain awater content of 10-20% or 5-10%, preferably 5-6%, 4-5%, or 3-4%, or2-3%, more preferably 1-2% or 0-1% (w/w).

Foam Drying

A preferred process of the invention involves subjecting thepreservation sample to such pressure and temperature conditions so thatthe sample begins to bubble, forming a foam.

The temperature within the preservation sample will be different fromthat external to the sample due to the endothermic nature of theevaporation process. References to temperature are to the conditionsexternal to the preservation sample, for instance, where a largeindustrial freeze dryer is used, to the temperature of the shelf. Thisusually corresponds to the freeze dryer temperature setting.

A preferred embodiment of the invention achieves this by reducing thepressure while maintaining temperature conditions. The pressure isadjusted to at or below 8, 7, 6, preferably 5, 4, 3, more preferably 2,1.5, 1, most preferably 0.8 or 0.5 mbar, while maintaining thetemperature setting at a temperature above 0° C., preferably of between10° C. to 15° C.; 15° C. to 20° C.; 20° C. to 25° C.; 25° C. to 30° C.;or 30° C. to 37° C. These conditions are maintained for at least 1, 2,3, 4, 5, 8, 10, 12, 16 or 24 hours.

Another embodiment of the invention achieves foam formation by changingthe temperature while maintaining reduced the pressure conditions. Thetemperature setting is increased to above 20° C., preferably to between20° C. and 30° C.; 30° C. and 40° C.; 40° C. and 50° C.; or 50° C. and70° C.; or the temperature setting is in the range of 10-50° C.,preferably 20-40° C., more preferably 25-35° C. Pressure conditions aremaintained at a reduced level of or below 8, 7, 6, preferably 5, 4, 3,more preferably 2, 1.5, 1, most preferably 0.8 or 0.5 mbar. Theseconditions are maintained for at least 1, 2, 3, 4, 5, 8, 10, 12, 16 or24 hours.

Removing Solvent to Form a Foamed Glass

A subsequent stage of the foam drying method of the invention involvesremoving solvent until the foam dries to form a solid. In one embodimentof the invention, this is achieved by maintaining the pressure andtemperature conditions at those applied in order to achieve foamformation. For instance, the pressure is maintained at or below 8, 7, 6,preferably 5, 4, 3, more preferably 2, 1.5, 1, most preferably 0.8 or0.5 mbar while maintaining the temperature setting at a temperatureabove 0° C., preferably between 2° C. and 10° C., 10° C. and 20° C.; 20°C. and 30° C.; 30° C. and 35° C., 35° C. and 40° C. most preferablybetween 5° C. and 25° C. These temperature and pressure conditions aremaintained for 1, 2, 3, 4, 5, 6, 8, 10, 12, 18 hours or more in order toobtain a solid with a solvent content less than or equal to 10, 8, 5, 4,preferably 3, 2 or most preferably 1% (w/w).

Another embodiment of the invention increases the temperature settingduring solvent removal to a higher temperature setting than thatmaintained earlier in the process. This advantageously allows thesolvent to leave the sample at a quicker rate so that the method of theinvention can be completed in a shorter time. For instance, thetemperature setting is increased to above 0° C., preferably between 2°C. and 10° C.; 10° C. and 20° C.; 20° C. and 30° C.; 30° C. and 40° C.;40° C. and 50° C.; 50° C. and 60° C. while maintaining the pressure ator below 8, 7, 6, preferably 5, 4, 3, more preferably 2, 1.5, 1, mostpreferably 0.8 or 0.5 mbar. These temperature and pressure conditionsare maintained for 1, 2, 3, 4, 5, 6, 8, 10, 12, 18 hours or more inorder to obtain a solid with less than 10, 8, 5, 4, preferably 3, 2 ormost preferably 1% (w/w) water content.

Another embodiment of the invention reduces the pressure setting duringsolvent removal to a lower pressure setting than that used during foamformation. This advantageously allows the solvent to leave the sample ata quicker rate so that the method of the invention can be completed in ashorter time. For instance, the pressure setting is decreased to at orbelow 5, 4, 3, preferably 2, 1, 0.8, more preferably 0.5, 0.1. mostpreferably 0.05 or 0.1 mbar, while maintaining the temperature at orabove 0° C., preferably between 10° C. and 20° C.; 20° C. and 30° C.;30° C. and 35° C. or above 40° C. These temperature and pressureconditions are maintained for 1, 2, 3, 4, 5, 6, 8, 10, 12, 18 hours ormore in order to obtain a solid with a solvent content less than orequal to 5, 4, preferably 3 or 2 or more preferably 1% (w/w).

Foam Drying Including a Freezing Step

The method of the invention optionally involves freezing the sample.Freezing the sample prior to foam drying has the advantage of increasedreproducibility between samples in a batch. This is due to all thesamples starting the process from the same physical condition of beingfrozen. The preservation samples may be wholly or partially frozen.

Freezing is optionally carried out before subjected the sample toreduced pressure by placing the preservation sample at a temperaturebelow 0° C. for a suitable amount of time to allow the sample to freeze.Preferably the temperature used is at or below −10° C., −15° C., −20°C., −30° C., −40° C., −70° C. or −140° C. The sample may be left at atemperature below 0° C. for 1, 2, 3, 4, 5, 8, 16 or more hours to allowfreezing to occur.

For some samples, particularly samples that are easily damaged bysolvent crystal formation such as cell preparations or other biologicalsystems, it is preferable to freeze the sample slowly at a rate of lessthan or equal to 0.1, 0.5, 1, 2, 3, 4, 5° C. per hour. Othercompositions are preserved more effectively by freezing instantaneously,for instance by snap freezing in liquid nitrogen. This method isparticularly beneficial for proteins or viral particles. Freezing byevaporation also results in rapid freezing of the sample.

Alternatively, the preservation sample is frozen by subjecting thesample to reduced pressure such that the sample becomes wholly orpartially frozen. Such quench freezing is carried out within a bulkfreeze dryer apparatus, at a shelf temperature of or above 0° C., 10°C., 15° C., 20° C., 30° C., 37° C. Preferably the shelf temperature isbetween 5 and 35° C., more preferably between 10 and 20° C., mostpreferably at 15° C. The pressure is optionally reduced initially to 200mbar for 5, 10, 20, 30, 60 minutes or more to allow degassing. In orderto freeze the sample, the pressure is reduced further to a pressureequal to or below 2, 1, 0.5, 0.2, 0.1 mbar. This pressure is maintainedfor at least 5, 10, 20 or 30 minutes until the sample is wholly orpartially frozen.

Subsequent steps of foam formation and removing solvent to form a solidare as described above.

In a preferred embodiment of the invention, the steps of freezing thesample within the freeze dryer and foam formation are performed at aconstant temperature, preferably altering the pressure conditions.

In a further preferred embodiment the steps of freezing the samplewithin the freeze dryer, foam formation and solvent removal to form asolid, are performed at a constant temperature, preferably altering thepressure conditions.

In a further embodiment of the invention, both pressure and temperatureconditions are different during the steps of freezing the sample, foamformation and solvent removal to form a solid.

The processes of the invention preferably use containers with a waterrepellent interior surface. This is achieved through coating theinterior surface with a hydrophobic composition, for instance bysiliconisation. Siliconisation is achieved by processes that are wellknown to those skilled in the art. Alternatively, the water repellentinterior surface is achieved by the container being made of a waterrepellent composition.

The presence of a water repellent interior surface of the containermakes foam formation more likely to occur and more reproducible. Thisallows lower polyol concentrations to be used in the preservation samplewhich in turn decreases the length of time necessary to dry the sample,reduces the effect of Maillard reactions or other harmful interactionsbetween the polyol and the active agent. Where the preservation samplescomprises a vaccine, the resultant solid is reconstituted quickly due tothe lower amount of polyol present and the resultant vaccine solution isless viscous, allowing easier administration.

Drying without Freezing or Foam Formation

A particularly preferred method of the invention involves drying IPV inthe presence of a stabilising agent, and preferably a bacterialpolysaccharide, using a gentle process that avoids exposure of PV tofreezing or foam formation so that IPV is subjected to less stressduring the drying process and a high degree of antigenicity is retained.

This method is particularly applicable for use where a lowerconcentration of the glass forming polyol, for example at concentrationbelow 10% (w/v), more preferably below 5% (w/v), is advantageous and ashorter drying time is preferred.

Loss of Solvent by Evaporation (Evaporative Drying—Step b)

The process of drying without freezing or foam formation involvessubjecting the preservation sample to such pressure and temperatureconditions so that the preservation sample looses solvent byevaporation, without the sample freezing or bubbling to form a foam.

The temperature within the preservation sample will, at times, bedifferent from that external to the sample due to the endothermic natureof the evaporation process. References to temperature are to theconditions external to the preservation sample, for instance, where alarge industrial freeze dryer is used, to the temperature of the shelf.This usually corresponds to the freeze dryer temperature setting.

Optionally a preliminary step of degassing the preservation sample ispresent in the method of the invention. The pressure is reduced to at orbelow 200 mBars, preferably between 200 and 35 mBars, for a period of atleast 5 minutes before the pressure is reduced further.

A preferred embodiment of the invention achieves evaporative drying byreducing the pressure while controlling the temperature conditions. Thepressure is adjusted to at or below 30, 25, 20, preferably 15, 12, mostpreferably 10, 8, 7, 6, 5, 4, 3, 2 or 1 mbar, while maintaining thetemperature setting at a temperature above 0° C., preferably of between4° C. to 37° C., 4° C. to 10° C., 10° C. to 15° C.; 15° C. to 20° C.;20° C. to 25° C.; 25° C. to 30° C.; or 30° C. to 37° C.; or 37° C. to45° C. These conditions are maintained for at least 1, 2, 3, 4, 5, 8,10, 12, 16 or 24 hours, preferably for between 2-4 hours, 4-6 hours, 6-8hours, 8-12 hours or 12-18 hours. In a particularly preferredembodiment, the pressure is maintained above 2 mbars where thetemperature setting is 15° C. in order to prevent freezing of thesample. In a preferred embodiment, the temperature is maintained at 15°C. and the pressure is set to between 5-10 mBars, more preferably 6-9mBars, most preferably around 8 mBars. Where a higher temperaturesetting is used, slightly lower pressure is possible without freezingthe sample and where a lower temperature setting is used, the pressureshould be maintained at the higher level to prevent freezing. Preferablythe conditions are maintained for a sufficient period of time so thatthe evaporation rate has slowed so that the temperature of the sample isapproximately the same as that external to the sample.

Preferably, the preservation sample should not freeze or boil to form afoam and looses solvent to form a viscous liquid or a highly viscousliquid.

Removing Solvent to Form a Highly Viscous Liquid

A subsequent stage of the method of the invention involves removingsolvent until the preservation sample dries to form a highly viscousliquid without foam formation and preferably without freezing.

In one embodiment of the invention, this is achieved by maintaining thepressure and temperature conditions at those applied in the firstevaporative drying stage. For instance, the pressure is maintained at orbelow at or below 30, 25, 20, preferably 15, 12, most preferably 10, 8,7, 6, 5, 4, 3, 2 or 1 mbar, while maintaining the temperature setting ata temperature above 0° C., preferably of between 5° C. to 37° C., 5° C.to 10° C., 10° C. to 15° C.; 15° C. to 20° C.; 20° C. to 25° C.; 25° C.to 30° C.; or 30° C. to 37° C. For a temperature setting of 15° C., apressure of 5-10 mBars, preferably 6-9 mBars, most preferably around 8mBars is maintained for between 4-24 hours, preferably 1-4, 4-8, 8-12 or12-16 hours. These temperature and pressure conditions are maintainedfor 1, 2, 3, 4, 5, 6, 8, 10, 12, 18 hours or more in order to obtain ahighly viscous liquid with a solvent content less than or equal to 15,12, preferably 10, 8, 5, 4, 3, 2 or 1% (w/w).

Another embodiment of the invention increases the temperature settingduring solvent removal to a higher temperature setting than thatmaintained earlier in the process. This allows the solvent to leave thesample at a quicker rate so that the method of the invention can becompleted in a shorter time. For instance, the temperature setting isincreased to above 0° C., more preferably above 20° C., preferablybetween 5° C. and 37° C., 5° C. and 10° C., 10° C. and 20° C.; 20° C.and 30° C.; more preferably 30° C. and 40° C.; more preferably 40° C.and 50° C.; most preferably 50° C. and 60° C. while maintaining thepressure at or below 30, 25, 20, preferably 15, 12, most preferably 10,8, 7, 6, 5, 4, 3, 2 or 1 mbar. These temperature and pressure conditionsare maintained for 1, 2, 3, 4, 5, 6, 8, 10, 12, 18 hours or more inorder to obtain a solid with less than or equal to 15, 12, preferably10, 8, 5, 4, 3, 2 or 1%. This embodiment requires the active agent to beheat stable at the temperature used for the method to be carried outsuccessfully.

A preferred embodiment of the invention reduces the pressure settingduring solvent removal (step c) to a lower pressure setting than thatused earlier in the process (step b). This allows the solvent to leavethe sample at a quicker rate so that the method of the invention can becompleted in a shorter time. It also enables a higher proportion of thesolvent to be lost. For instance, the pressure setting is set to at orbelow 7, 6, preferably 5, 4, 3, more preferably 2, 1.5, 1, mostpreferably 0.8, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, or 0.005 mbar, whilemaintaining the temperature at or above 0° C., preferably between 10° C.and 20° C.; 20° C. and 30° C.; 30° C. and 35° C. or above 40° C. Thesetemperature and pressure conditions are maintained for 1, 2, 3, 4, 5, 6,8, 10, 12 or 18 hours or more in order to obtain a solid with a solventcontent less than or equal to 15, 12, preferably 10, 8, 5, 4, 3, 2 or 1%(w/w) preferably as determined by Karl Fischer coulometric moistureanalyser (Eur. J. Pharm. Biopharm. (2000) 50; 277-284).

Preferably, steps b) and c) should be completed in a time equal to orless than 18 hours, more preferably 16, 14, 12, most preferably 10, 8, 6or 4 hours.

A dried composition is a composition from which solvent has been removedby evaporation, boiling, or sublimation leaving a solvent content lessthan or equal to 15, 12, 10, more preferably 8, 5, 4, 3, 2 or 1% (w/w),preferably as determined by the Karl Fischer method. Preferred ranges ofsolvent content are 1-3%, 3-5%, 5-10% or 10-15% (w/w). The term includeshighly viscous liquids as well as dried foamed glass and lyophilisedsolids.

A highly viscous liquid is defined as a material from which solvent hasbeen removed by evaporation without boiling, leaving a solvent contentless than or equal to 15, 12, 10, preferably 8, 5, 4, 3, 2 or 1% (w/w),preferably as determined by the Karl Fischer method. Preferred ranges ofsolvent content are 1-3%, 3-5%, 5-10% or 10-15% (w/w). The highlyviscous liquid has a sufficiently low solvent content such that theactive agent is preserved in a stable state for at least 3,6,9,12 or 24months at 4° C., allowing the active agent to retain at least 40, 50,60, preferably 70, 80, 90 or 95% of its activity and/or antigenicityover this period. Preferably, the highly viscous liquid has a solidappearance but is a glass and is able to flow very slowly over a periodof 2, 4, or 6 days, more preferably 1, 2, 3, 4, 6, 7, 10 or 12 months.The extremely slow flow may be measured by inverting a receptaclecontaining the highly viscous liquid and leaving at room temperatureuntil the highly viscous liquid is observed to flow. In a preferredembodiment, the highly viscous liquid will not appear to flow after 2, 4or 6 days, preferably 2, 3 or 4 weeks, more preferably 2, 4, 6, 8, 10 or12 months in an inverted position.

A viscous liquid is defined as the product of the primary phase ofsolvent removal, at the end of which the majority of solvent has beenlost from the sample. This point can be recognised because the rate ofevaporation slows down so that the temperature of the sample returns tothe ambient temperature as the endothermic effect of bulk evaporation islost.

A foamed glass is a dried composition containing a glass forming polyol,which is formed by a method wherein the preservation sample is subjectedto such temperature and pressure conditions that the sample bubblesvigorously or boils so that a foam is formed as the sample dries.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

EXAMPLES

The examples below are carried our using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Evaporative Freezing Process

The process was carried out using a Heto Drywinner 8-85 freeze-dryer inwhich shelf temperature maybe regulated to within 1° C., the finaltemperature of the condenser is −85° C., pressure is regulated with ableed valve and 6 thermocouples are available to measure the producttemperature.

A preservation sample was made by adding a stabilising agent (either 10%trehalose or 3.5% sucrose) and an active agent to an aqueous solution.Samples were put into the freeze dryer with a shelf temperaturemaintained at a fixed temperature setting of 15° C. throughout theprocess. The pressure was initially reduced to 200 mBar and maintainedat this level for 10 minutes before reducing the pressure further. At1.5 mBar, the solutions began to freeze due to evaporative cooling asshown in FIG. 1. The pressure is further reduced to 0.1 mBar to allowthe samples to become fully frozen. The pressure was then increased tobetween 0.8 mBar and 3.5 mBar at which point a foam formed as water waslost from the sample. Under the conditions of the experiment, no boilingwas seen in a control sample containing only water. The samples may beloosing water through evaporation rather than through boiling. After 18hours under these conditions, the samples are dried and the foamedsolution becomes a foamed glass.

A similar process was successfully performed keeping the shelftemperature at other temperature settings up to 37° C.

Example 2 Establishment of Freezing Conditions

Samples were made by dissolving sucrose in water to give 1%, 5%, 10% and20% solutions. Samples were put into the freeze dryer with a shelftemperature maintained at 15° C. throughout the process. The pressurewas initially reduced to 200 mBar and maintained at this level for 10minutes before reducing the pressure further to 50 mBars, 5 mBars, 2.5mBars, 0.75 mBars, 0.4 mBars and 0.2 mBars. Each pressure level wasmaintained for 20 minutes to allow the temperature to equilibrate andthe temperature of the sample was read using a thermocouple.Thermocouples were attached to samples with different sucroseconcentrations and the temperatures recorded in table 1 are mean valuesof the temperatures.

Results

All samples froze between 1.66 and 1.11 mbars, irrespective of theconcentration of sucrose present. The temperatures measured at differentpressures were very close to those predicted from the triple pointcurve. Therefore the presence of sucrose does not appear to have a largeeffect on the temperature of the samples at different pressures.

In a preferred method of the invention, it is necessary to avoidfreezing of the sample. This could be achieved by maintaining thepressure above 2 mBars using a shelf temperature of 15° C. At lowertemperatures the pressure should be maintained at a higher level whereasuse of a higher temperature would allow the pressure to be reducedfurther without the samples freezing.

TABLE 1 Measured Theoretical Liquid/ Pressure temperature temperaturefrozen 1000 mBar 15° C. liquid 50 mBar 15° C. liquid 5 mBar 1° C. 1° C.liquid 2.5 mBar −5° C. −7° C. liquid 0.75 mBar −21° C. −21° C. frozen0.4 mBar −22° C. −27° C. frozen 0.2 mBar −27° C. −32° C. frozen

Example 3 Foaming Conditions for Samples with Different SugarConcentrations

Preservation samples containing 0%, 5%, 10%, 15%, 20%, 25% and 50%sucrose were made. Samples were put into the freeze dryer with a shelftemperature maintained at 15° C. throughout the process. The pressurewas initially reduced to 200 mbars and maintained at this level for 10minutes before reducing the pressure further. The pressure was furtherreduced to 0.1 mbars to allow the samples to become fully frozen. Thepressure was then increased to either 0.788 mbars, 0.812 mbars or 3.5mbars in subsequent experiment These conditions were maintained for 3hours for the 3.5 mbars and 0.812 mbars experiments and for 6 hours forthe 0.788 mbars experiment. The physical characteristics of each samplewere evaluated.

Results

As shown in table 2, at a pressure of 3.5 mbars, a high sucroseconcentration of 50% was required for reliable formation of foam. Incontrast, a lower pressure of 0.8 mbars allowed reliable foam formationat lower sucrose concentrations of 10-25%. The use of lower sucroseconcentration could be advantageous for preserved samples to be used invaccines for instance. Therefore a process using 0.8 mbars and a 10-25%sucrose content is preferred.

TABLE 2 Pressure % sucrose Physical characteristics  3.5 mbars 20 ⅘foamed, ⅕ viscous liquid  3.5 mbars 25 ⅖ foamed, ⅗ viscous liquid  3.5mbars 50 5/5 foamed 0.812 mbars 5 Ring of crystallisation and bubbles0.812 mbars 10 All foamed 0.812 mbars 15 All foamed 0.812 mbars 20 Allfoamed 0.812 mbars 25 All foamed 0.788 mbars 5 Ring of crystallisationand bubbles 0.788 mbars 20 All foamed 0.788 mbars 25 All foamed 0.788mbars 50 Foam and syrup

Example 4 The Effect of Using Siliconized Containers

Preservation samples containing 5%, 10%, 15% and 25% sucrose were madeand added to vials, some of which were siliconized. In one experiment,samples were put into the freeze dryer with a shelf temperaturemaintained at 15° C. throughout the process. The pressure was initiallyreduced to 200 mbars and maintained at this level for 10 minutes beforereducing the pressure further. The pressure was further reduced to 2.8mbars for 3 hours. During this period, the pressure fell to 2.00 mbarsas the presence of water vapour decreased. The physical characteristicsof each sample were evaluated.

In a second experiment, samples were put into the freeze dryer with ashelf temperature maintained at 37° C. throughout the process. Thepressure was initially reduced to 200 mbars and maintained at this levelfor 10 minutes before reducing the pressure further. The pressure wasfurther reduced to 2.4 mbars for 3 hours. During this period, thepressure fell to 1.06 mbars as the presence of water vapour decreased.The physical characteristics of each sample were evaluated.

Results

Siliconization had an effect on the degassing of the samples. Thereduction of pressure to 200 mbars resulted in degassing of samples insiliconized vials but not in unsiliconized vials. Degassing was seen bybubbling of the sample.

The siliconisation of the vial also made foam formation more likely tooccur and more reproducible (table 3). Siliconisation of vials allowsfoam formation to occur reproducibly at lower polyol concentrations. Thelower polyol concentration decreases the length of time necessary to drythe sample and reduces the effect of Maillard reactions or otherinteractions with the polyol harming the active agent. Where the sampleinvolved is a vaccine, this reduces the viscosity of the sample andallows easier administration.

TABLE 3 Temperature and Characteristics Characteristics pressure %sucrose nonsiliconised vial siliconised vial 15° C., 2.8 mbars 5%Viscous fluid 15° C., 2.8 mbars 10% Viscous fluid foamed 15° C., 2.8mbars 15% Viscous fluid 15° C., 2.8 mbars 25% Viscous fluid 37° C., 2.4mbars 5% 3 viscous fluid 2 foamed 37° C., 2.4 mbars 10% All viscousfluid 5 foamed 1 viscous fluid 37° C., 2.4 mbars 15% All foamed 37° C.,2.4 mbars 25% All foamed

Example 5 Comparison of Preservation of Hib-IPV by Conventional FreezeDrying or by Foam Drying

The active agent to be preserved was a mixture of the PRP polysaccharideof Haemophilus influenzae b (Hib) and three strains of inactivated poliovirus (IPV). The preservation sample was made by dissolving Hib-IPV ineither a 3.15% sucrose solution or a 10% trehalose solution.

The samples were lyophilised either by using a conventional freezedrying sample that required three days to perform in a large freezedryer, or by using the foam drying method described in example 1.

The samples were reconstituted in water and an ELISA was used to assessthe retention of antigenicity of the three polio virus strains. Threepolyclonal antibodies and three monoclonals, one against each strain,were used in separate ELISAs. Results are presented as a percentage ofthe reading given for a sample which had not undergone the freeze dryingor foam drying procedure.

The preserved samples are assessed for their immunogenicity in vivo byinoculating groups of ten mice with the reconstituted IPV-Hib,withdrawing blood from the mice and monitoring levels of antibodiesagainst IPV and Hib polysaccharides, for instance by ELISA or Westernblotting. The degree of protection is assessed in a challenge mousemodel.

Results

Using either sucrose or trehalose as the polyol, the antigenicity of IPVwas maintained better using the foam drying technique compared to usingconventional freeze drying.

TABLE 4 ELISA - type 1/2/3 % Method of drying Polyol content PolyclonalMonoclonal Freeze drying 3.15% sucrose 46/49/58* 25/0/0 Foam drying3.15% sucrose 85/97/106 55/68/57 Freeze drying 10% trehalose 47/43/58Foam drying 10% trehalose 93/86/84 72/75/87 *The experiment freezedrying in the presence of 3.15% sucrose was repeated five times and theresults shown are from one representative experiment.

Example 6 Protective Effect of Freeze Drying IPV in the Presence of HibPolysaccharides

Preservation samples were prepared containing 3.15% sucrose and IPV or amixture of IPV and Hib polysaccharides. The samples were inserted into aHeto Drywinner 8-85 freeze-dryer and freeze dried at a temperaturesetting of −32° C. for 40 hours followed by continued drying at 4° C.for 16 hours.

The samples were reconstituted in water and an ELISA was used to assessthe retention of antigenicity of the three polio virus strains. Threemonoclonal antibodies, one against each strain, were used in separateELISAs to assess the degree of antigen retention in the reconstituted,freeze dried sample compared to a reference sample that had not beenfrozen. Results are presented as a percentage of the reading given for asample which had not undergone the freeze drying or foam dryingprocedure.

Results

As shown in table 5, the presence of Hib polysaccharide in thepreservation sample with IPV, led to greater retention of IPV antigensafter freeze drying than that achieved when IPV was freeze dried alone.The Hib polysaccharides have a preserving effect on IPV antigenicity inaddition to that achieved by having sucrose present as a stabilisingagent.

TABLE 5 Composition freeze dried Polyol content ELISA - type 1/2/3 % IPV3.15% sucrose 26/25/0 IPV-Hib 3.15% sucrose 52/68/0

Example 7 Effect of Different Stabilising Agents on Freeze DryingIPV-Hib

Preservations samples were made containing IPV-Hib and using either3.15% sucrose; 2.5% sorbitol, 0.8% glutamine and 0.01% HSA; MMRstabiliser and lactose; 3% glycine, 2% arginine and 4% sucrose; or 4%sucrose and 2% glycine as stabilising agent. The experiment included asample with 3.15% sucrose as stabilising agent using double theconcentration of IPV-Hib. The samples were freeze dried using aconventional three day freeze drying cycle in a batch freeze dryer.

The samples were reconstituted in water and an ELISA was used to assessthe retention of antigenicity of the three polio virus strains. Threepolyclonal antibodies and three monoclonals, one against each strain,were used in separate ELISAs. Results are presented as a percentage ofthe reading given for a sample which had not undergone the freeze dryingor foam drying procedure.

The preserved samples are assessed for their immunogenicity in vivo byinoculating groups of ten mice with the reconstituted IPV-Hib,withdrawing blood from the mice and monitoring levels of antibodiesagainst IPV and Hib polysaccharides, for instance by ELISA or Westernblotting. The degree of protection is assessed in a challenge mousemodel.

Results

Increasing the dose of IPV from 40/8/32 DU/dose to 80/16/64 DU/dose ledto an increase in retention of antigenicity of IPV as shown in table 6.Variation in the stabilising agent also influenced retention of antigenswith 4% sucrose/2% glycine and 2.5% sorbitol/0.8% glutamine/0.01% HASproducing higher retention of antigens as shown by ELISA data.

TABLE 6 Polyclonal Monoclonal Stabilising agent ELISA results ELISAresults 3.15% sucrose 50/50/70 25/0/0 2.5% sorbitol 55/72/72 33/50/00.8% glutamine 0.01% HSA MMR stabiliser 59/62/65 28/25/0 lactose 3.15%sucrose 84/92/120 102/138/0 Double dose of IPV-Hib 3% glycine 2%arginine 4% sucrose 4% sucrose 46/62/78 25/50/15 2% glycine

Example 8 Reproducibility of Sample Quality after Freeze Drying, FoamDrying or Foam Drying with a Freezing Step

Preservation samples are made up comprising IPV, mumps, measles,rubella, varicella zoster virus, CMV, hepatitis, HSV1, HSV2, respiratorysyncitial virus, dengue, paramyxoviridae such as parainfluenza,togaviridae and influenza viruses, and/or Hib as the active agent. Theactive agent is dissolved in an aqueous solution containing a polyol.Multiple samples are preserved by either freeze drying, foam dryingusing a freezing step following the protocol described in example 1, orfoam drying without a freezing step using a protocol described inexample 4. Samples are reconstituted in an aqueous solution and theiractivity assessed. This is accomplished using ELISA assays as describedin example 5 using antibodies specific to native antigens. In the caseof live viruses, the titre of each sample is established by using thevirus to infect suitable host cells and assessing the infectivity byplaque formation or by immunocytochemistry. Where immunogeniccompositions or vaccines are foam dried, the retention of immunogenicitycan be tested in an animal model by immunising groups of animals withvaccine which is foam dried or freeze dried and boosting the immuneresponse for instance at 14 and 28 days after the first immunisation.Serum is isolated from animals at the end of the immunisation scheduleand its titre against the vaccine is tested using standard assays, forinstance by ELISA, immunocytochemistry, Western blotting,immunoprecipitation, serum bacteriocidal assay or agglutination assay.Results are complied, first by comparing the activity of the activeagent after freeze drying, foam drying with a freezing step, or foamdrying without a freezing step. Secondly, the degree of reproducibilityof the preservation technique is assessed by comparing the range ofactivities after subjecting samples to each of the three preservationmethods.

Example 9 Long Term Storage of Active Agents Preserved by Freeze Drying,and Foam Drying

Preservation samples are made up comprising IPV, mumps, measles,rubella, varicella zoster virus, CMV, hepatitis, HSV1, HSV2, respiratorysyncitial virus, dengue, paramyxoviridae such as parainfluenza,togaviridae and influenza viruses, and/or Hib as the active agent. Theactive agent is dissolved in an aqueous solution containing a polyol.Multiple samples are preserved by either freeze drying, foam dryingusing a freezing step following the protocol described in example 1, orfoam drying without a freezing step using a protocol described inexample 4. Samples are aged by storing at 37° C. or 23° C. for sevendays and are compared for activity with samples that have been keep at4° C. Samples are reconstituted in an aqueous solution and theiractivity assessed. This will be accomplished using ELISA assays asdescribed in example 5 using antibodies specific to native antigens. Inthe case of live viruses, the titre of each sample is established byusing the virus to infect suitable host cells and assessing theinfectivity by plaque formation or by immunocytochemistry. Results arecomplied, first by comparing the activity of the active agent afterstorage at elevated temperatures with storage at 4° C. Secondly, thedegree of reproducibility of the preservation technique is assessed bycomparing the range of activities after subjecting samples to each setof conditions.

Example 10 Method for Drying without Freezing or Foam Formation

Preservation samples containing 5%, 10%, 15% and 25% sucrose were madeand added to vials. Samples were put into a freeze dryer at atemperature setting of 15° C. throughout the process. The pressure wasinitially reduced to 200 mBars and maintained at this level for 10minutes to allow degassing before reducing the pressure further. Thepressure was further reduced to 8 mbars for two to three hours duringwhich time thermocouples inside the samples showed that the sampletemperature reduced to 4° C. due to evaporative cooling. After 2-3hours, the temperature of the samples returned to 15° C., indicatingthat evaporation under these temperature and pressure conditions wasnear completion. During this stage of the process, the sample did notboil to form a foam or freeze so that an active agent within the sampleis exposed to as little stress as possible. The samples have a solidappearance, similar to the final product.

Further drying of the samples was achieved by reducing the pressurefurther to 0.1 mbars while keeping the shelf temperature setting at 15°C. These conditions were maintained for a further 10-16 hours. Duringthis phase, the sample temperature remained at 15° C. since the rate ofevaporation was slow. Further drying took place and the resultant samplehad a solid appearance. If the sample was place on its side, the samplecontents slowed very slowly, over a period of days or months showingthat the sample is a liquid glass of high viscosity. FIG. 2 shows thatthe containing holding the highly viscous liquid can be inverted withoutprovoking immediate flow of the highly viscous liquid.

Example 11 Retention of IPV Immunogenicity after Drying without Freezingor Foam Formation

Samples dried according to the method of example 10 have not beensubjected to stresses associated with the bubbling that accompanies foamformation or freezing. Experiments were performed to determine whetherthis method produced a high level of antigen retention when used to dryIPV.

Three separate experiments were performed in which IPV was resuspendedin an aqueous solution with 10% sucrose or 10% trehalose as thestabilising agent. The samples were put into siliconised vials whichwere placed into a Heto Drywinner 8-85 freeze-dryer and the temperaturewas set to 15° C. The pressure was initially reduced to 35 mBars todegas the sample. After 10 minutes, the pressure was further reduced to8 mBars and was kept at this level for two hours. During this period thetemperature setting was kept at 15° C. and the temperature into thesample was monitored. As water evaporated from the sample, thetemperature dropped to 4° C. but towards the end of the two hours, thetemperature returned to 15° C. as the rate of evaporation slowed. Nobubbling or foam formation occurred under these conditions. The pressurewas then reduced further to 0.1 mbars and these conditions weremaintained for 16 hours more in the first two experiments and for 10hours more in the third experiment.

The samples were reconstituted in water and an ELISA was used to assessthe retention of antigenicity of the three polio virus strains. Themonoclonal antibody against type 3 IPV, was used in an ELISA to assessthe degree of antigen retention in the reconstituted, freeze driedsample compared to a reference sample that had not been frozen. Resultsare presented as a percentage of the reading given for a sample whichhad not undergone a drying procedure.

Results

The dried samples had a solid appearance however they appeared to be inthe form of a highly viscous liquid/glass since, over a period of days,the dried solid was able to flow if the container was inverted at roomtemperature.

TABLE 7 Retention of type 3 IPV antigen as determined by ELISA using amonoclonal antibody (drying without foaming or freezing) 1^(st)experiment 2^(nd) experiment 3^(rd) experiment Formulation (18 hourcycle) (18 hour cycle) (12 hour cycle) 10% sucrose 75% 78% 91% 10%trehalose 82% 79% 93%

These levels of type 3 IPV antigen retention compares very favourablywith the freeze drying results shown below where very low values wereusually found in the same ELISA format when a monoclonal antibodyagainst type 3 was used.

TABLE 8 Retention of type 1, 2 and 3 IPV antigens as determined by ELISAusing a monoclonal and polyclonal antibodies (freeze drying) ELISA -type 1/2/3 % Method of drying Polyol content Polyclonal MonoclonalFreeze drying 3.15% sucrose 46/49/58* 19/25/0 Freeze drying 10%trehalose 47/43/58  25/0/0 *The experiment freeze drying in the presenceof 3.15% sucrose was repeated five times and the results shown are fromone representative experiment.

Example 12 Long Term Storage stability of Dried IPV Stored as a HighlyViscous Liquid/Glass

IPV dried using the method described in Example 11 was stored at 4° C.for 9 months. The samples were reconstituted in water with 150 mM NaCland an ELISA was used to assess the retention of antigenicity of thethree polio virus strains. Three monoclonal antibodies, one against eachstrain, were used in separate ELISAs to assess the degree of antigenretention in the reconstituted stored sample. A similar ELISA had beencarried out on reconstituted samples from the same batch prior tostorage. All results were compared to a reference sample that had notbeen dried. Results are presented as a percentage of the reading givenfor a sample which had not undergone a drying procedure.

Results

TABLE 9 Retention of IPV antigens after storage as a highly viscousliquid for 9 months Treatment Type 1 ELISA Type 2 ELISA Type 3 ELISADried/reconstituted 72% 75% 88% Not stored Dried/reconstituted 70% 94%90% 9 months 4° C.

Therefore IPV which has been dried by the method described in Example 11can be stored at 4° C. for at least 9 months without loss ofantigenicity.

Example 13 Comparison of the Immunogenicity in vivo of IPV after Dryingto Form a Highly Viscous Liquid and Reconstitution Compared to UndriedIPV

Groups of 10 Wistar rats were inoculated with various dilutions of IPVwhich had been dried in the presence of 10% sucrose to form a highlyviscous liquid using the method disclosed in Example 10 andreconstituted. Further groups of 10 Wistar rats were inoculated withreference samples of IPV which had been prepared in the same way butwhich had not been dried.

After 21 days, sera were taken from all the rats and the sera weretested in separate immunoprecipitation assays using Type 1, Type 2 andType 3 polio virus.

Results are shown in table 10 that contains: —a) the number of responantrats for each IPV dilution, b) the ED50 which is the dose that isrequired to ensure that 50% of the rats seroconvert as assessed by theimmunoprecipitation assay and c) the relative potency of the dried andreconstituted IPV compared to the undried reference IPV.

TABLE 10 Immunogenicity of IPV after drying to form a high viscosityliquid (JLE017/05) and reconstitution compared to an undried referenceIPV (JLE097) Number RP of respondant relative Sample undiluted 1/1.251/3.125 1/7.81 ED50 potency JLEO17/05 Type 1 10 9 6 5 6.37 0.956 Type 26 4 3 3 7.14 0.825 Type 3 6 8 2 1 18.18 1.051 JLE097 Type 1 10 10 10 73.33 1.120 Type 2 8 6 5 2 3.12 0.951 Type 3 7 6 4 1 16.91 1.172Reference Type 1 10 8 4 6.37 Type 2 7 5 2 2.93 Type 3 5 3 0 22.57

JLEO17/05 is a IPV batch that was dried to form a highly viscous liquidand subsequently reconstituted. JLE097 is the undried reference.

Table 10 shows that the number of respondants inoculated with eachdilution of IPV is similar between the two batches of dried andreconstituted IPV and the undried reference sample. In general, Type 1IPV elicited the best immune response, with Type 2 eliciting an immuneresponse in slightly fewer rats. Type 3 elicited the weakest immuneresponse.

The process of drying to form a highly viscous liquid does not impairthe ability of IPV to elicit immunoprecipitating antibodies in vivo. Arelative potency (RP) reading of 1.0 indicates that the sample elicitsan equivalent response to the reference sample. Both dried samplesproduce RP readings of close to 1.0 for all three types of polio virusindicating the drying process does not effect the ability of the sampleto elicit an immune response.

Example 14 Effect of Drying to form a Highly Viscose Liquid UsingSucrose or Trehalose as Stabilising Agent on the Ability of IPV toElicit an Immunoprecipitating Immune Response In Vivo

Groups of 10 Wistar rats were inoculated with IPV which had been driedin the presence of either 10% sucrose or 10% trehalose as described inExample 2, and then reconstituted. Further groups of 10 Wistar rats wereinoculated with an equivalent amount of IPV that had not been dried, asreference samples.

After 21 days, sera were collected from all rats and animmunoneutralisation assay, as described in Example 5 was used to assessthe amount of immunoneutralising antibody that had been raised againsteach of Type 1,Type 2 and Type 3 polio virus.

Relative potencies were calculated for each sample by comparing theimmune response to that elicited by the undried reference sample.

Results are shown in Table 11.

TABLE 11 Comparison of drying in sucrose and trehalose Relative potencyLot in vivo Type 1/ Humidity % Duration Number Sugar present Type 2/Type3 Karl Fischer (hours) Jle017 10% trehalose 0.95/0.82/1.05 nd 7 31CO3/0110% sucrose 0.69/1.20/0.97 4.6% 18 31CO3/02 10% trehalose 0.60/0.94/0.9 11.5% 18 03D02/01 10% sucrose 0.74/1.05/0.96 5.9% 12 03D02/02 10%trehalose 0.58/0.98/1.06 10.6% 12

The amount of water remaining in samples was lower when sucrose was usedas stabilising agent with approximately 5% humidity remaining comparedto approximately 10% when trehalose was used as the stabilising agentmeasured by the Karl Fischer method.

Both sucrose and trehalose were effective at stabilising IPV during thedrying process so that the reconstituted IPV gave relative potencyreadings approaching 1.0 for most of the different types of polio virus.The relative potencies were particularly good for Type 3 polio viruswhich looses its immunogenicity relatively easily.

Example 15 Measurement of Humidity by Karl Fischer

Analysis was carried out in a Karl Fischer titrometer (Aqua30.00—Elektrochemie Halle). The sample was weighed out and placed intothe oven at a setting of 80° C. The sample was flushed with nitrogen gasand then added to hydranal reagent (Riedel de Hahn) in order to performthe analysis by coulometry.

The invention claimed is:
 1. An immunogenic composition comprising: (a)inactivated polio virus (IPV) comprising type 1, type 2, and type 3polio virus, (b) a capsular polysaccharide or oligosaccharide antigenfrom Haemophilus influenzae b, and (c) a stabilizing agent, allformulated as a dried composition, which after reconstitution is capableof generating an immune response against polio virus, wherein theantigenicity of type 1, type 2, and type 3 polio virus is retained at alevel of at least 40% of the antigenicity of a reference sample whichhas not been formulated as a dried composition, and wherein thestabilizing agent comprises a glass-forming polyol selected from thegroup consisting of: trehalose, sucrose, sorbitol, raffinose, mannitol,lactose, lactitol, or palatinit.
 2. The immunogenic composition of claim1, wherein the polysaccharide or oligosaccharide is conjugated to acarrier protein.
 3. The immunogenic composition of claim 2, wherein thepolysaccharide or oligosaccharide is conjugated to tetanus toxoid. 4.The immunogenic composition of claim 1, wherein the polysaccharide oroligosaccharide is adsorbed onto aluminium phosphate.
 5. The immunogeniccomposition of claim 1, further comprising phenol red.
 6. Theimmunogenic composition of claim 1, wherein the dried composition isfreeze dried.
 7. The immunogenic composition of claim 1, wherein thedried composition is a foamed glass.
 8. The immunogenic composition ofclaim 1, wherein the dried composition is a highly viscous liquid. 9.The immunogenic composition of claim 8, wherein the highly viscousliquid has not been frozen.
 10. A kit comprising the immunogeniccomposition of claim 1, in one container and liquid acellular or wholecell diphtheria, tetanus and Bordetella pertussis (DTP) vaccine in asecond container.
 11. The kit of claim 10, further comprising HepatitisB surface antigen in the second container.
 12. A vaccine comprising theimmunogenic composition of claim
 1. 13. A container with a waterrepellent internal surface containing the vaccine of claim 12.